Molecular Characterization Of Single Cells and Cell Populations For Non-Invasive Diagnostics

ABSTRACT

The invention discloses diagnostic techniques based on single cell genomics, consisting of obtaining a blood sample, enriching a sub-population of cells present in the blood sample, sequestering individual cells or group of cells from the blood sample, obtaining sequencing data from the sequestered cells or group of cells, using genetic variant information to determine the provenance of the cells, and genetically analyzing the cells of the correct provenance to provide a diagnostic readout. Using the cell-based testing techniques of the invention, the number of false positives is greatly reduced when compared to cell-free DNA (cfDNA) based traditional testing techniques. The invention may be effectively employed for non-invasive prenatal (NIPT) diagnostics, oncological testing and other diagnostic procedures.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 62/142,663, filed on Apr. 3, 2015, which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the fields of non-invasive prenatal testing(NIPT), single cell genomics based testing procedures, and non-invasivediagnostic testing in oncology.

BACKGROUND ART Non-Invasive Pre-Natal Diagnostics

Non-invasive pre-natal diagnostics are a growing area of development, inpart due to increasing parental age (and therefore genetic risk) and thepresence of genetic abnormalities in a significant percentage of theinfant population. The tests currently on the market, Ariosa, Verify andXX from Verinata, Natera and Sequenom respectively rely on the detectionof key abnormalities via counts of molecules derived from differentchromosomes in the blood of the pregnant mother, and enable detection ofTrisomies 21, 18 and 13.

While these tests have gained relatively widespread market adoption,they are limited to being marketed as a ‘screening’ test due to theirrelatively low positive predictive value (PPV). As an example, one suchscreening test, when testing for Trisomy 18, has true positive rate ofabout 40 patients in 400 positive screening tests. That translates toabout a 10% PPV. Because of the potential for false positives, apositive result using the non-invasive test needs to be followed up byan amniocentesis confirmatory test that has a much lower error rate (andhigher PPV).

The high false positive rate (and difficulty in developing a truediagnostic) is due to the fact that fetal cell-free DNA (fcfDNA) ispresent in concentrations of only 4-12% as compared to the mother'scell-free DNA (cfDNA). Consequently, for a fetal trisomy (say Trisomy18), if there are 3 as opposed to 2 copies of chromosome 18, the changein a sample that is 5% fetal DNA is only (5%/2)=2.5%. The copy numberdetermination therefore needs to be very accurate, as data has a greaterthan 5% spread and statistical methods need to be employed to determineany imbalance while sequencing at a high depth of coverage. There are anumber of different commercial tests based on cfDNA and this type ofconsideration.

While previous tests have been proposed based on fetal cells in themother's blood, they have been hard to develop into commercial testsbecause of the variable cell number recovered and the relatively lowconcentration of fetal cells to maternal cells in the final sample. Anumber of different modalities have been used to separate fetal cellsout using either posts in a microfluidic channel, macro scaleimmunomagnetic separation, size (Isolation by Size of Tumor cells i.e.ISET) or ferromagnetic properties of red blood cells. No one modalityhas demonstrated fetal cell purities of above 10% post enrichment, andthereby low purities have precluded commercial development. For a morethorough treatment of these and related topics, the reader is referredto U.S. Pat. No. 8,058,056, U.S. Pat. No. 8,293,524 and U.S. PatentApplication Publication No. 2013/0017538 A1.

Single Cell Analysis

In areas unrelated to prenatal diagnostics, methods and devices havebeen proposed to enable the analysis of single cells. The simplestapproach, termed Limiting Dilution, consists of measuring theconcentration of cells in a certain volume, followed by mixing of thewhole sample and dispensing a volume that is expected to contain asingle cell in each of many wells. This has the limitation of resultingin 1 cell on average per well, and a distribution of 0, 1, and 2 cellswith a few higher numbers, but where only about 60% of wells contain asingle cell in optimized protocols. Another related approach is toutilize a fluorescence activated cell sorter (FACS) instrument in orderto sort single cells directly into wells of a well plate. This approachsuffers from some of the same limitations in success rate, and requiresexpensive equipment to perform.

A number of different approaches for isolating and analyzing singlecells using microfluidic devices have also been proposed. One approachpreviously commercialized by Fluidigm, Inc. as part of their C1 productoffering utilizes a trap within the flow of a microfluidic channel, andmicrochannel based valve-ing to isolate the cell and to perform lysis,followed by nucleic acid amplification and extraction for up to 96cells.

One approach previously proposed for single cell immobilization was touse an array of lateral junctions. The approach provided for a method ofeither patch clamp recording or electroporation of single cells, whichwould lead to lysis, but did not clearly describe a method for furtheranalyzing the resulting nucleic acids from single cells by performingthe necessary amplification, sequestration and extraction of theamplified material. For a more rigorous treatment of this approach, thereader is referred to U.S. Pat. No. 8,058,056. The single cellimmobilization methods described may also be followed up by microscopybased detection of chromosome abnormalities for each cell.

OBJECTS OF THE INVENTION

In view of the limitations of the prior art, it is an object of theinvention to provide a non-invasive diagnostic method based on singlecell genomics that results in significantly lower false positives ascompared to the traditional techniques based on analyzing cfDNA ofpopulation of cells.

It is another object of the invention to provide a high-reliability andhigh-sensitivity testing protocol for NIPT, oncology and otherdiagnostic procedures, that has a much higher positive predictive value(PPV) than possible through the techniques of the prior art.

It is yet another object of the invention to provide for non-invasive,prenatal testing techniques that can produce highly accurate diagnosticreadouts of aneuploidies and other genetic diseases of the fetus.

It is another object of the invention to provide for non-invasivetesting techniques that can produce highly accurate diagnostic readoutsto assist in prognostics and determination of treatment efficacy inoncology.

Still other objects and advantages of the invention will become apparentupon reading the detailed specification and reviewing the accompanyingdrawing figures.

SUMMARY OF THE INVENTION

The objects and advantages of the invention are secured by an apparatusand methods of performing diagnostics based on single cell genomics. Thediagnostics techniques provided by the invention are well suited forNon-Invasive Pre-natal Testing (NIPT), oncological testing, or otherdiagnostics procedures.

A blood sample is first obtained from an expecting mother or a patient.Then a sub-population of cells from the blood sample is enriched toachieve a higher level of purity. The sub-population can be fetal cellsfor NIPT usage, or cancer cells for oncological testing. The enrichmentprocess may employ a variety of techniques including immunomagneticseparation, microfluidic manipulation, cell morphology based separation,anti-body binding based separation, or other enrichment techniquesavailable to a person of average skill. These techniques may be employedsingly or in combination to achieve the desired levels of purity,ideally greater than or equal to 10% against the background.

After enrichment, individual cells or groups/subsets of cells of thesub-population are isolated/separated/sequestered. A number oftechniques may be employed for cell separation or sequestration. Theseinclude trapping individual cells at junctions of microfluidic channelsthat have differing sizes. Preferably, cell trapping happens at channeljunctions at the bottom of the wells of a well plate. Cell trappingusually occurs under the influence of negative pressure applied to thesmaller channel of such a junction. Selective releasing and retrappingof cells can also be used to enrich certain cell populations based onmorphology.

Once the cells have been trapped, a lysing solution is preferablyapplied to the trapped/immobilized cells. The lysing solution can betransported into the wells by pipetting or by using another microfluidicchannel. Eventually, the lysed contents of the individual cell or groupsof cells from the enriched sub-population are obtained inseparate/isolated reservoirs or wells for further processing.

At this stage, various pressure manipulation techniques may be usedaround the microfluidic structures to minimize or cut off unwanted flowin selected channels. Now nucleic amplification of the cellular contentsis preferably performed using a variety of available techniques,including Polymerase Chain Reaction (PCR) and Ligase Chain Reaction(LCR). Furthermore, individual cells may advantageously be barcoded inthe reservoirs such that sequencing occurs only on barcoded cells.

At this point, sequencing is performed on the amplified (and preferablybarcoded) lysed cells. The sequence data is obtained and recorded, basedon which the provenance of the separated cells is determined. Cellprovenance is preferably determined based on a genetic variantinformation, such a single nucleotide variant (SNV) information. Cellprovenance may reveal that in the case of NIPT, the cell(s) is/are ofmaternal origin or of fetal origin. In the case of oncological testing,cell provenance may reveal that the cell(s) is/are normal i.e. ofpatient origin or of cancer/tumor origin. Cell provenance mayalternatively be determined by identifying individual molecules of thecells by unique barcodes, and then applying a consensus operation.

Sequence data is also used to determine the genetic characteristics ofthe cells applicable for the particular diagnostics of interest. Howeverthe genetic characteristics only need to be determined for cells thathave the correct origin i.e. fetal cells for NIPT, and tumor cells foroncology.

For NIPT, the fetal genetic characteristics may indicate the presence ofan aneuploidy such as a particular trisomy, or the presence of anotherSNV related disorder such as an addition or a deletion of a nucleotide,or the presence of still some other inherited disease of the fetus. Ofcourse, the genetic characteristics may also reveal that the fetus isnormal. Finally, a diagnostic readout is provided for the mother and/orthe fetus.

For oncological testing, the genetic traits may indicate the presence ofa particular genetic mutation, or a cancer. Of course, the geneticcharacteristics may reveal the absence of any such disorder from thepatient. Based on the above findings, an appropriate cancer therapy orother prognostic measures may be recommended for the patient. In asimilar fashion, these techniques may be used for diagnosticapplications related to the characterization of rare immune cells, orauto-immune diseases, or organ transplant rejection.

The invention admits of its applicability to other diagnosticdisciplines besides NIPT and oncology. Clearly, the techniques andmethods of the invention find many advantageous embodiments. The detailsof the invention, including its preferred embodiments, are presented inthe below detailed description with reference to the appended drawingfigures.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 shows an exemplary workflow for non-invasive genetic testingbased on cell-based diagnostics, according to the present invention.

FIG. 2A-B show a comparison of the testing protocols of the traditionalcell-free DNA (cfDNA) based detection of fetal abnormalities (trisomy),and cell-based diagnostics using cell isolation by limiting dilution,according to the teachings of the present invention.

FIG. 3A shows cell immobilization at microfluidic channel junctions,specifically, single cells trapped at junctions and lysed by introducinga buffer such that cellular contents end up in secondary wells.

FIG. 3B shows a cross-sectional view of one portion of the microfluidicarchitecture for the embodiment of FIG. 3A.

FIG. 3C shows a three dimensional side view of an exemplary pressingsubstrate for the embodiment of FIG. 3A-B.

FIG. 4A shows a variation where a microfluidic channel delivers the cellsuspension directly to the wells where trapping happens. Cells are thenlysed by adding lysis buffer to the wells either by pipetting, or viaanother microfluidic channel.

FIG. 4B shows a cross-sectional view of one portion of the microfluidicarchitecture of FIG. 4A.

FIG. 5A shows, based on mock data, chromosome 21 copy number variationdata for normal versus maternal cell-free DNA (cfDNA) using traditionaltechniques, where the fetus is a carrier of Trisomy 21. Fetal DNAcontent found physiologically is varied along the x-axis, from 10-0%.

FIG. 5B shows, based on mock data, chromosome 21 copy number variationdata based on cell-based sequences of normal maternal cells versusmaternal cells where the fetus is a carrier of Trisomy 21, according tothe invention.

DETAILED DESCRIPTION

The figures and the following description relate to preferredembodiments of the present invention by way of illustration only. Itshould be noted that from the following discussion, alternativeembodiments of the structures and methods disclosed herein will bereadily recognized as viable alternatives that may be employed withoutdeparting from the principles of the claimed invention.

Reference will now be made in detail to several embodiments of thepresent invention(s), examples of which are illustrated in theaccompanying figures. It is noted that wherever practicable, similar orlike reference numbers may be used in the figures and may indicatesimilar or like functionality. The figures depict embodiments of thepresent invention for purposes of illustration only. One skilled in theart will readily recognize from the following description thatalternative embodiments of the structures and methods illustrated hereinmay be employed without departing from the principles of the inventiondescribed herein.

The various aspects of the invention will be best understood byinitially referring to the exemplary workflow presented in FIG. 1embodying the main aspects of the invention. According to the diagnostictechniques of the invention, a blood sample is first obtained from apatient—step 10. Then a specific sub-population of cells in the bloodsample is enriched—step 20. Cells of the sub-population are thenisolated or sequestered into individual cells or groups of cells—step30. The isolated cell(s) is/are then sequenced—step 40. Sequencing istypically performed after first performing any necessary nucleic acidamplification.

The sequence data thus obtained is then analyzed for geneticcharacteristics/traits related to a particular condition ordisease/disorder—step 50. Finally a diagnostic readout is provided—step60. Importantly, the analysis/diagnosis is carried out only for thecells of the correct provenance, so that the number of false positivesin the diagnostic readout is significantly reduced. This is possiblebecause as a part of the analysis of the sequence data of the cells, theprovenance of the cells of the sub-population is determined, and thusthe diagnosis is carried out only for the cells of the correct type ofprovenance.

The techniques presented above are particularly suited for performingnon-invasive prenatal tests (NIPT), as well as for performingnon-invasive tests in oncology, transplant rejection, auto-immunedisease, and rare immune cell characterization. Explained further, theblood sample from an expecting mother or an oncology patient may beanalyzed using the above process. In such an analysis, circulating fetalcells (CFCs) or circulating tumor cells (CTCs) in the blood sample areanalyzed for the diagnosis of birth defects in the fetus or the presenceof cancer. The process is further illustrated in the diagram of FIG.2A-B in the context of NIPT, against comparison with the traditionalmethods.

Specifically, in FIG. 2A representative of cell-free DNA (cfDNA) basedanalysis of the prior art, cfDNA is extracted from the plasma of theblood of the patient (expecting mother in the case of NIPT). cfDNAsample thus obtained, consists of the cfDNA of maternal origin shown byreference numerals 52 as well as the fetal cell-free DNA (fcfDNA) offetal origin—also sometimes referred to as cell-free fetal DNA (cffDNA),shown by reference numeral 54 and represented by the dashed lines ofchromosomal shapes.

The combined maternal and fetal cell-free DNA is then sequenced by a DNASequencer 56, which is then followed by a diagnostic analysis phase 58.Diagnostic analysis 58 of the prior art is based on the sequencing ofthe entire blood sample, consisting of both cfDNA and fcfDNA. Thesequencing typically required to be done is at a fairly high depth,typically greater than 1,000× or greater than 10,000×. Based on thesequencing step, the proportion/percentage of the cells of fetal DNA areestimated in the cfDNA sample.

Then based on this percentage, a diagnostic evaluation of the fetal DNAis provided which may include an indication of the presence of ananeuploidy in the fetal DNA. This technique of the prior art is prone toa high number of false positives. In other words, the diagnosticevaluation only serves as a screening test that needs to be followed byusually invasive confirmatory test(s).

In contrast, as shown in FIG. 2B representative of the cell-baseddiagnostic evaluation techniques of the present invention, fetal cellsin the blood sample of the mother are first enriched. The blood samplewith enriched sub-population of fetal cells is shown as a collection ofthe individual cells of the mother 102, and those of the fetus 104. Inparticular, three such maternal cells are shown by reference numerals102 and one fetal cell is shown by the reference numeral 104 in FIG. 2B.

The step of cell enrichment is followed by cell separation orsequestration as mentioned above. During this step, individual cells orgroups of cells are separated or sequestered. After cell separation, thepopulation of fetal cells analyzed may be further enriched by onlyanalyzing cells that have a higher probability of being fetal asdetermined by either morphological factors, immunohistochemistry orstaining of specific proteins expressed by the target fetal cells.

FIG. 2B shows three wells 106 containing maternal cells 102, and onewell 108 containing a fetal cell 104. After sequestration, cellsequencing can commence as shown in FIG. 2B. Because each sequenceoriginates with a single DNA strand per cell, each cell sample accordingto the invention may be sequenced at a relatively low depth (i.e. of theorder of 50×). The low-depth sequencing is usually accompanied by aconsensus operation to determine the correct provenance of the cells aswill be further explained below.

This is a major advantage over the techniques of the prior art thatrequire cfDNA samples to be sequenced at depths greater than 1,000× or10,000×. However, it is worth noting that with many more single cellsequencing samples, the overall number of reads would typically becomparable and thus the sequencing cost is likely to be on parity ascompared to traditional techniques. Nonetheless, the invention admits ofhaving this differentiation potentially leading to reduction indiagnostic costs with the use of appropriate technological means anddevices.

The fetal sequence obtained above is shown by the dashed box 112 in FIG.2B, while the maternal sequences are shown by solid boxes 110. Asalready mentioned above, the present techniques allow for a much lowerfalse positive rates in the diagnostic readout than otherwise possibleby the techniques of the prior art (FIG. 2A). Thus such a test can serveas a screening and a confirmatory test, without necessarily requiring afollow-up confirmatory, and usually invasive, test as in the currentlyavailable testing modalities of the prior art.

Having explained the basic operation of the diagnostic system andmethods of the invention, let us know return our attention to theworkflow/protocol of FIG. 1, and look at steps 20-60 of that workflow inmuch more detail.

Enrichment:

Although the invention admits of any cell enrichment techniques that maybe employed in the workflow of FIG. 1, of particular interest areimmunomagnetic separation, used preferably in conjunction with antibodybinding markers and cell morphology based separation criteria such ascell size, cell deformability/elasticity, dielectrophoresis, inertialforces and shear forces, etc. According to the invention, these methodsmay be used singly or in combination. But first let us provide a briefintroduction to microfluidics, as its various structures and devices areutilized by various aspects of the present invention.

Microfluidics is a multidisciplinary field with applications in manyindustries including biotechnology. It is the field of study where lowvolumes of fluids are processed for cell manipulation, multiplexing,high-throughput screening and automation. Microfluidic systems haveshown unique advantages in performing functions such as controlledtransportation, immobilization, and manipulation of cells, as well asseparation, mixing, and dilution of chemical reagents, which enables theanalysis of intracellular parameters, even on a single-cell level.

The present invention applies the above benefits of microfluidics in itsvarious embodiments. Specifically, in one embodiment, the enrichmentprocess may use immunomagnetic separation. Immunomagnetic separation(IMS) methods are based on the attachment of small magnetizableparticles/beads to cells via antibodies or lectins. When the mixedpopulation of cells is placed in the influence of a magnetic field,those cells that have beads attached are attracted/repelled to/from themagnet and may thus be separated from the unlabeled cells.

Thus the enrichment process as employed in the invention may utilize oneor more cell markers to which magnetic beads may be attached, and thenpositive and negative fractions of the mixed cell population can besorted utilizing a magnetic field. This may be accomplished whileutilizing a variety of microfluidic devices. The principles and workingsof these techniques are well understood in the art and will not bedelved into detail in this specification. Briefly, magnetic beads arefunctionalized to bind different cell populations. In one instance,beads may be pre-conjugated with one or more antibodies to specificproteins, such that the conjugated beads recognize and bindpreferentially to cells expressing antigens to said antibodies. Theseare then brought into contact with the starting cell population, wherebyonly a subset of cells bind with the beads. This is referred to as thedirect binding method.

Alternatively, in what is called the indirect binding method, antibodieswith a linker molecule (like biotin or immunoglobulin) are first addedto the cell suspension and bind the antigen. The cells are then exposedto beads coated with the complementary linker molecule (likestreptavidin) in order to bind beads to cells expressing the antigen ofinterest. The cell mixture is then exposed to a magnetic field eitherinside a cavity, or while flowing across a field inside a capillary ormicrofluidic channel. In this manner, bead decorated cells are separatedfrom the rest of the suspension (i.e. from cells that do not express theantigen of interest).

In another embodiment, the enrichment process may use one of the variouscell morphology or physical property criteria such as cell size, shape,elasticity/deformability, inertial forces, or dielectric constants.These techniques can also be typically used in conjunction with variousmicrofluidic devices. The principles and workings of these techniquesare also well understood in the art and will not be delved into detailin this specification. Generally a cell mixture is introduced into aseparation cavity, which may be a microfluidic channel, and is thensubjected to forces that discriminate between cell populations based onphysical properties. Examples include size exclusion, inertial forcesunder flow, and shear forces. The separated populations are thencollected either by traditional fluid handling (i.e. pipetting) or bydirecting flow of the different populations into different microfluidicchannel outlets, or a combination of the above.

Other embodiments use any antibody binding characteristics of the cellsin the blood sample as markers to be exploited during cell enrichment.Still other embodiments may use any other techniques known to a personof average skill in the art in the enrichment step of FIG. 1, includingFluorescence Activated Cell Sorting (FACS). As already stated, all theabove techniques during the step of enrichment may be used singly or anyin combination to achieve the desired level of purity of the enrichedcell population.

The desired level of purity should be at least 1% but ideally 10% ormore of fetal cells against a background of maternal cells for NIPTdiagnostics. Similarly, the desired level of purity should be at least1% but ideally 10% or more of tumor or other types of diseased cellsagainst a background of normal cells for oncological or otherdiagnostics.

Now let us return to the workflow of FIG. 1 and consider the next step30 of cell sequestration/separation in much more detail.

Cell Sequestration:

After enriching the cells of a sub-population of interest, such as fetalcells for NIPT procedures and cancer cells for oncology procedures,individual cells or groups of cells of the sub-population areseparated/sequestered. The present invention takes advantage of thevarious possible modalities of cell sequestration.

In one variation, single cell separation/sequestration may be performedby Limiting Dilution into wells preferably aimed at dispensing one cellper well of a well plate as shown in FIG. 2B. This step results in astatistical distribution of mostly 0, 1, or 2 cells per well.Alternatively, a FACS system may be use to both enrich the desired cellpopulation (as mentioned in the above section), and to dispense singlecells per well.

Additionally, a variety of microfluidic architectures and devices may beused. One such microfluidic architecture utilized in a preferredembodiment uses single cells that get trapped at the junction of smalland large microfluidic channels.

After cell trapping, the sub-population of target cells (e.g. fetalcells for NIPT, and cancer cells for oncology) that are analyzed may befurther enriched by only analyzing cells that have a higher probabilityof being the target cells as determined by either morphological factors,immunohistochemistry or staining of specific proteins expressed by thetarget cells. The cells are then lysed with an appropriate lysing agent,and then the contents of each lysed cell are passed into a separatereservoir. This process is illustrated in the microfluidic apparatus 200and its associated methods of FIG. 3A according to the invention.

FIG. 3A shows a view of microfluidic apparatus 200 as seen from the toplooking down, or from the bottom looking up. The figure shows a largemicrofluidic channel 206, connected with a smaller microfluidic channel204A. Microfluidic channel 206 contains an enriched cell suspensionobtained after the enrichment process described above. Individual cellsin the cell suspension in channel 206 are shown by small circles.According to the invention, a pressure differential between the smallermicrofluidic channel 204A and larger microfluidic channel 206 results inindividual cells in the suspension to get trapped at the junction ofmicrofluidic channels 206 and 204A. A cell 220A trapped at the junctionof channels 206 and 204A is shown in FIG. 3.

The same setup is replicated thrice in the arrangement shown in FIG. 3Awith corresponding elements 204B-C, 220B-C indicated, and channel 206extending across all three. However, where convenient in the belowexplanation, we may facilitate the discussion by employing only thefirst portion of the above scheme, with the knowledge that same/similarprocesses occur in the other replicated portions, and will drawattention to any differences if and when necessary.

To facilitate understanding, FIG. 3B further shows a cross-sectionalview of the first portion of apparatus 200. Cell 220A is seen trapped inFIG. 3A-B at the junction of channels 206 and 204A and is thusimmobilized. Such trapping/immobilization will occur under the influenceof a negative pressure in channel 204A. In other words, and as will beapparent to skilled artisans familiar with an intuitive application ofthe basic laws of physics and fluid dynamics, if a negative pressuredifferential between channel 204A and channel 206 is created, that willencourage cells in the solution carried by channel 206 to be trapped atits junctions with smaller channel 204A.

As already mentioned, after cell trapping, the sub-population of targetcells (e.g. fetal cells for NIPT, and cancer cells for oncology) thatare analyzed may be further enriched by only analyzing cells that have ahigher probability of being the target cells as determined by eithermorphological factors, immunohistochemistry or staining of specificproteins expressed by the target cells. Specifically, cells that aredetermined to be of non-target origin (e.g. maternal cells for NIPT, andnormal cells for oncology), may be removed from the traps by applyingpositive pressure to wells 212A-C, in cases where imaging determinescells 220A-C to be likely of non-target origin. In this way, celltrapping and selective release may be used to enrich a certain rare cellpopulation.

At this stage, a lysing agent/buffer can be introduced in microfluidicchannel 206 at an inflow 208 and exited at an outflow 210. The lysingagent will lyse cells 220A, 220B, 220C and their lysed contents willthen travel under the applied pressure and be deposited in separatereservoirs 212A, 212B and 212C respectively.

The pressure differential between channels 206 and 204A as describedabove, can be easily created by pressing a mating surface from the topagainst the upper structure of well plate 201. As shown in FIG. 3A-B,each channel junction of channels 206 and 204A-C is surrounded by thewalls of reservoirs 202A-C in the shape of squares with rounded corners.An exemplary structure is that of a Society for Biomolecular Screening(SBS) format well plate with a rigid upper structure formed of injectionmolded plastic.

When an appropriate surface with interfaces mating to this rigid upperstructure is pressed against it, this results in pressure applied tochannels 204A-C and consequently to the inputs of reservoirs/wells212A-C. It is under the influence of this pressure that cell trappingoccurs as described above. Additionally, inflow 208 and outflow 210 mayin turn be connected to input and output wells of the same upperstructure (not shown) for ingress or egress of various fluids, includinglysing buffer. Additionally, any other appropriate mechanisms known tothose skilled in the art may be used to cause negative pressure in thedesired channel(s), e.g. by using a syringe for suction.

In summary, pressure manipulation in various channels of FIG. 3A-B maybe achieved by pressing a sheet/substrate of glass or some othersuitable material as needed against the microfluidic structures fromabove or below. One such pressing substrate 214 is shown in thecross-sectional view of FIG. 3B. Notice that when pressing substrate 214is pressed upwards, the three protrusions in pressing substrate 214 willpress against the various microfluidic channels, thereby increasing theflow resistance or entirely cutting it off. Specifically, as shown inFIG. 3B, once cell 220A has been lysed and its contents have depositedin reservoir/well 212A, pressing substrate 214 may be pushed upwards tocut off flow in channel 204A thereby ensuring that lysed contents ofcell 220A in well 212A are not contaminated. Such a technique is alsoreferred to as a “foot on the hose valve-ing” technique.

In a similar fashion, and advantageously, after the introduction of thelysing agent in channel 206, each of trapped/immobilized cells 220A-Care kept separate from each other by having an increased flow resistancein channel 206 between each of its junctions with channels 204A-C of thethree replicated portions shown in FIG. 3A. This can be accomplished bypressing a sheet/substrate of glass or some other suitable materialagainst channel 206 from below to reduce or cut-off flow selectively.

In one embodiment, this is accomplished as follows. Notice threereservoirs indicated by reference numerals 202A, 202B and 202C in thereplicated architecture on a well plate 201 shown in FIG. 3A. Eachreservoir 202A, 202B, 202C has a familiar outline as shown by thesquares with rounded corners. Analogously to the foot-on-the hosevalving scenario for channel 204A described above in reference to FIG.3B, pressing substrate 214 may have protrusions to cut off flow inchannel 206 at its intersections with the left and right edges ofreservoirs 202A-C and also to cut off flow in channels 204A-C at theirintersections with the lower edges of reservoirs 202A-C. Such anexemplary pressing substrate 214 is further shown in a three dimensionalside view in FIG. 3C, which has protrusions in the shape of two squares215A and 215B that when pressed against two reservoirs, will cut offflow entirely around them.

Once the lysed contents of cells 220A-C have been deposited and securedin reservoirs/wells 212A-C, sequencing and analysis can begin as will befurther taught below. Note that wells 212A-C are again reminiscent of astandard well plate as will be observed by the skilled reader. Such adesign facilitates integration of this process with standard fluidhandling and/or Quantitative Polymerase Chain Reaction (qPCR) equipment.The architecture shown in FIG. 3 also has the advantage of havingminimal cross-contamination (“cross-talk”) due to diffusion betweendifferent reservoirs 212A, 212B and 212C.

The above scheme can be used to sequester contents of individual cells220A-C or of multiple cells from the enriched suspension of cells inchannels 206. Multiple cells may be trapped and lysed from the enrichedsuspension with their contents stored in reservoirs 212A-C. This may beaccomplished by repeating the application of negative pressure after acell 220A, 220B, 220C has been trapped and lysed, so that the sameprocess can be repeated for additional cells of the enriched suspension.

Another embodiment of the invention uses cell trapping at channeljunction residing inside or at the bottom of the wells of a standardwell plate. In such a scenario a smaller microfluidic channel connectsto a well at a junction where the cell trapping happens. This scenariois illustrated in the microfluidic apparatus 250 and its associatedmethods of FIG. 4A, according to the invention.

FIG. 4A shows a microfluidic channel 260 feeding a reservoir 254 havinga well 255 caused by a punch-hole in reservoir 254. Notice that incontrast to the embodiment of FIG. 3A, here we are drawing a distinctionbetween the surrounding reservoir and the actual well in it caused by apunch-hole, and where the channel junction resides. Reservoir 254 andwell 255 are a part of a standard well plate, a section 252 of which isshown. Enriched cell solution is fed from channel 260 to reservoir 254.

Under the influence of a negative pressure, as in the embodiment of FIG.3, cell trapping occurs inside well 255 at its bottom with its junctionto a smaller channel 256. Specifically, a cell 270 is being showntrapped at the intersection of well 255 and channel 256. Note that thesame scheme is replicated in FIG. 4A, however repeating referencenumerals have been omitting from the drawing and the associatedexplanation for clarity and to avoid undue repetition. To facilitateunderstanding, FIG. 4B further shows a cross-sectional view of the firstportion of apparatus 250.

As with the embodiment of FIG. 3A-C, after cell trapping, the populationof fetal cells (or cells of interest) analyzed may be further enrichedby only analyzing cells that have a higher probability of being thetarget cells, as determined by either morphological factors,immunohistochemistry or staining of specific proteins expressed bytarget cells. Cells that are determined to be of non-target origin maybe removed from the traps under pressure. Referring to FIG. 4A-B, ifimaging determines cell 270 to be of likely non-target origin, then itmay be removed by applying negative pressure to reservoir 254. In thisway, cell trapping and selective release may be used to enrich a certainrare cell population prior to lysis.

Once cell 270 is trapped and immobilized, a lysing buffer/solution,followed by a DNA amplification solution or other agents/reagents asdesired, can be added directly to reservoir 254 by a pipette.Alternatively, the lysing buffer/solution can be added via anothermicrofluidic channel carrying the solution directly to wells 255. Such achannel 258 for carrying lysing and other/or solutions is shown in FIG.4A-B and is connected to reservoir 254 at punch-hole/well 255 via asmall channel 256.

Note that after lysing, channel 258 may be reused for carrying otheragents/reagents, such as eluting and DNA amplification solutions, forexample. Preferably, these reagents may be first carried by a mainchannel 251 which in turn may be fed by other feeding wells (not shown)that are selectively activated/pressurized as and when desired to feedthe above mentioned agents/reagents into reservoir 254. In an exemplaryscenario, main channel 251 will feed channel 258 that will in turn feedreservoir 254.

A person skilled in the art will recognize the vast variety ofmicrofluidic designs and options available to practice the principlesand teachings taught herein. The architecture shown in FIG. 4A-B alsohas the advantage of having a minimum cross-contamination (“cross-talk”)due to diffusion between different reservoirs 254 and wells 255.

Furthermore, and similarly to the architecture of FIG. 3, thearchitecture in FIG. 4 can be fully or partially influenced/covered by asheet/substrate of glass or some other suitable material as needed tomanipulate pressure, under the influence of which cell trapping at wells255 occurs, or to manipulate flow in various channels as desired. FIG.4B shows an exemplary pressing substrate 262 which when pressed upagainst the microfluidic structures above, will cut off flow in channel256 so that the lysed contents of cell 270 in well 255 stay secure.

As in the earlier embodiment, the above scheme can be used to sequestercontents of an individual cell 270 or of multiple such cells from theenriched cell solution. In either of the above techniques of cellsequestration, typically a group or subset of cells ranging from 1 to500 cells may be sequestered for further analysis.

Another variation uses Limiting Dilution Analysis for cellsequestration. Those skilled in the art will understand that LimitingDilution Analysis is a technique in cell biology for estimating thefrequency of a specific type of cell in a complex mixture of cells. Thistype of cell may be identified by its response to an activation signal.The activation signal can induce cell proliferation, differentiation, orthe expression of specific cellular functions in the responder cells,which may include cytotoxic activity or the release of cytokines orantibodies. As already described earlier, in this variation, single cellseparation/sequestration may be performed by Limiting Dilution intowells meant to preferably dispense one cell per well of a well plate asshown in FIG. 2B. This step typically results in a statisticaldistribution of mostly 0, 1, or 2 cells per well.

Still another variation employs Fluorescence Activated Cell Sorting(FACS) for cell separation/sequestration. FACS provides a technique forsorting a heterogeneous mixture of cells into two or more containers,one cell at a time. This sorting is based upon the specific lightscattering and fluorescent characteristics of each cell. Both the abovevariations may also employ the benefits of appropriate microfluidicsystems and architectures to achieve their desired objectives of cellsequestration, and obtain individual cells or a group of cells of asub-population of interest into isolated reservoirs/wells for furtheranalysis.

Now let us return to the workflow/protocol of FIG. 1 and consider thenext steps of 40, 50 and 60 of cell sequencing, analysis and diagnosisin much more detail.

Cell Sequencing and Genetic Analysis:

According to the above teachings, the contents of individual cells or agroup of cells of a sub-population of interest can be saved inreservoirs/wells. The cells of interest comprising the sub-populationcan be of fetal origin for NIPT diagnostics, or they can be tumor cellsfor oncological diagnosis, or they can belong to another disease ordisorder for the appropriate diagnosis.

At this point, any necessary nucleic acid amplification may be carriedout to the cell populations in the reservoirs/well. The invention isagnostic of such amplification methods available in the art. Anon-exhaustive list of such methods is Polymerase Chain Reaction (PCR),Ligase Chain Reaction (LCR), Loop Mediated Isothermal Amplification(LAMP), Nucleic Acid Sequence Based Amplification (NASBA), StrandDisplacement Amplification (SDA), Multiple Displacement Amplification(MDA), Rolling Circle Amplification (RCA), Helicase DependentAmplification (HDA), Ramification Amplification Method (RAM), etc.

After the necessary amplification, sequencing of the cell populations inthe reservoirs can begin with the intent of performing diagnosis.Preferably, prior to sequencing, a barcode is added to each cellularcontent in the reservoirs. Then sequencing needs to be performed only onthe barcoded contents. Sequencing or genetic sequencing, maps/sequencesthe genetic code of the cells in the reservoirs. According to theinvention, the diagnosis is based on the genetic characteristicsidentified in the gene sequences, and needs to be performed on the cellsthat were sequestered as explained above, and those which can bedetermined to be of the right type of provenance. Exemplary provenancetypes can be maternal or fetal origin in the case of NIPT, or normal orcancer origin in the case of oncological testing.

In other words, the genetic analysis to determine abnormalities needs tobe performed on cellular contents of only those reservoirs/wells of FIG.3A and FIG. 4A, that are determined to be of the correct provenance aswill be further taught below. Still further explained, and referring toFIG. 3A, sequence data from all reservoirs/wells will be first analyzedto determine the provenance of the cellular contents in eachreservoir/well. If for example, the cellular contents of reservoir 212Bin FIG. 3B are determined to be of fetal origin in NIPT, then only thecontents of reservoir 212B need to be further analyzed, and not those ofreservoirs 212A and 212C.

Thus for NIPT purposes, the analyzed cells would belong to the CFCs fromthe blood sample of the expecting mother. The diagnostic readout in thiscase will be based on the features of the fetal genome detected from thesequence data, uncontaminated by maternal DNA, and may include anindication of aneuploidies, such as trisomy 8, trisomy 9, trisomy 13,trisomy 18, trisomy 21, trisomy 22, an XXX status, an XXY status, an XYYstatus, etc. The diagnostic readout may also include an indication ofthe presence of other abnormalities such as insertions and deletions ofsingle nucleotide variants, or the presence of still other inheriteddiseases as indicated by the genetic sequence of the fetal cells.

Analogously, for oncological testing, the genetic analysis of thesequestered cell or group of cells needs to be performed only on thecell(s) that are known to be of cancerous origin, and hence would belongto the CTCs from the blood sample of the patient. The diagnostic readoutin this case will be based on the features of the genetic mutations andthe cancer genome detected from the sequence data of the tumor cells,and may include guidance on suitable therapies or other prognosticmeasures for the patient. The therapeutic guidance may includealterations to existing therapies or recommendations on entirely newones. In a similar fashion, these techniques may be used for diagnosticapplications related to the characterization of rare immune cells, orauto-immune diseases, or organ transplant rejection.

Now let us see how the provenance of the sequestered cells is properlyascertained prior to the above explained genetic analysis, according tothe invention. For this purpose, the invention checks single nucleotidevariants (SNVs) information of the cells from their sequence data. Thisis done for each reservoir/well i.e. 212A-C of FIG. 3A and 254/255 andtheir replicated counterparts of FIG. 4A. For NIPT purposes, based onthe differences in SNVs and other individual-specific geneticdifferences between maternal and fetal cells, the provenance of thecells is ascertained. For oncological diagnosis, based on thedifferences in SNVs and other genetic abnormalities of tumor cells thatare not present in normal cells (e.g. copy number variations, genefusions, etc.), the provenance of the cells is ascertained. Thediagnostic readout is then provided based only on the fetal cells in theformer case and based only on the tumor cells in the latter case. Itwill be clear to the skilled reader that either DNA or RNA/geneexpression may be used to determine cell provenance.

In an alternative variation, a consensus operation is employed to checkthe origin/provenance of each cell. In this variation, when applied toNIPT diagnostics, individual sequestered cells in each reservoir arefirst tagged by unique barcodes, which may be randomly generatedbarcodes. Then each molecular sequence is aligned to astandard/reference genome, and the results compared against each otherand then clustered into sets of like cells (reservoirs displayingminimal differences).

Based on a suitable consensus cutoff, then the majority of ‘like’sequences in the cluster are selected to belong to the mother(majority), and the remaining sequences are then known to belong to thefetus (minority), noting that some additional minority clusters may becontamination. NIPT genetic analysis is then performed on the molecularsequences of fetal origin, and a diagnostic readout provided asexplained above.

The above determination may also benefit from techniques to reduce theerrors present in the sequences of each starting molecule that issequenced (ideally before the assignment to the maternal or fetal set isperformed). In this variation, in addition to using cellular barcodes,unique molecular barcodes are attached to each starting molecule beforeamplification and sequencing. Thus sequences that originated from thesame individual starting molecular barcode must match exactly, onceerrors have been eliminated. In one embodiment, error elimination may bedone by an appropriate statistical approach such as outlier detection,or by one of a number of consensus operations like selecting only thealteration present in most sequences from the same molecular barcode.

Additionally, any other statistical approach may also be employed todetermine consensus amongst the sequences and to eliminate errors, andto consequently improve the determination of cell provenance. Theadvantage of the above consensus operation is that by its very nature,it serves to greatly reduce the effects of nucleic amplification andsequencing errors by requiring consensus from all sequences derived froma unique starting molecule. Another advantage of such a consensusprocess among different cell sequences is that the sequencing operationof the fetal cells may be carried out at much lower depths, of the orderof 50×.

Still another key advantage of the apparatus and methods of theinvention is that the number of false positives in the event diagnosticreadout is significantly reduced. This is further illustrated in FIG. 5Aand FIG. 5B, representing the effectiveness of the present inventionagainst standard techniques for diagnosing an aneuploidy such as trisomy21 based on mock data.

Specifically, FIG. 5A shows mock data based on cell-free DNA (cfDNA) ofa normal maternal blood sample represented by dark solid lines versusmaternal blood sample where the fetus is a carrier of Trisomy 21represented by dark dashed lines. The fetal DNA content foundphysiologically is varied along the x-axis, from concentrations of 10%to 0%. As fetal DNA concentration is changed from: 10% to 5% to 3% to0%, the corresponding change as compared to normal in the number ofchromosome 21 copies in maternal cfDNA sample where the fetus is thecarrier of Trisomy 21 is: 1/20=5% to 0.5/20=2.5% to 0.3/20=1.5% to0/20=0% respectively. This is amply illustrated by the minute change inlength of the dashed lines from left to right with the correspondingchange in fetal DNA content in FIG. 5A. Note that at 10% concentration,the fetal DNA content (3 copies of chromosome 21) would be 3/20=15%.

In contrast, in FIG. 5B representative of the cell-based non-invasivediagnostic techniques of the instant invention, normalized chromosome 21readouts of single cell sequences of normal maternal cells versusmaternal cells where the fetus is a carrier of Trisomy 21 are shown assolid and dashed dark lines respectively. The diagram illustrates thespike in the chromosome 21 readout for fetal cells against normalmaternal cells. The provenance of each cell read is indicated below thex-axis in FIG. 5B. Obviously the normalized maternal readout is 1.0 forthe number of chromosome 21 copies, corresponding to 2 such chromosome21 copies in the maternal cells—same as normal. However, the normalizedfetal readout is 1.5, corresponding to 3 such chromosome copies in thefetal cells with the trisomy—50% more than normal.

That is a difference in the signal of 50% as compared to 5% cfDNAmeasurements (also see FIG. 5A), a significant confirmatory differenceagainst the traditional non-invasive analysis of cfDNA shown in FIG. 5A.Unsurprisingly, the resultant signal-to-noise ratio (SNR) is also muchbetter than the traditional techniques. The noise level in cell-freetechniques is likely to be 5% versus 5-10% using the cell-basedtechniques of the instant invention because of the lowered sequencingdepth of coverage as explained above.

Thus the SNR of the present invention is likely be in the range of50%/10%-50%/5%=5-10 versus 5%/5%=1. That is an improvement in SNR of500-1000% over the techniques of the prior art. The gain in SNR resultsin much better sensitivity/specificity in pre-natal non-invasive tests,translating into a much better positive predictive value (PPV). Thisfurther gives the present techniques the ability to assay new fetalgenomic characteristics/traits like SNV related disorders.

Still in other variations, single cell immobilization using microfluidicdevices explained above (see FIG. 3A-C, and FIG. 4A-B and the associatedexplanation) may be followed by staining of the immobilized cells. Thestained cells can then be microscopically analyzed for detection ofchromosome abnormalities for each individual cell. This method has thepotential of lowering the cost while maintaining a high PPV of the testsas described above.

In view of the above teaching, a person skilled in the art willrecognize that the teachings and methods of present invention can beembodied in many different ways in addition to those described withoutdeparting from the principles of the invention. Therefore, the scope ofthe invention should be judged in view of the appended claims and theirlegal equivalents.

We claim:
 1. A cell-based diagnostic method, comprising the steps of:(a) enriching a sub-population of cells of a blood sample; (b)sequestering one or more cells of said blood sample; (c) sequencing saidone or more cells; (d) determining a provenance of said one or morecells based on said sequencing; (e) determining a genetic characteristicof said one or more cells based on said sequencing and said provenance;and (f) providing a diagnosis based on said genetic characteristic. 2.The cell-based diagnostic method of claim 1, further utilizing at leastone element selected from the group consisting of an immunomagneticseparation, a microfluidic manipulation, a cell morphology basedseparation and an antibody binding based separation, in said step (a) ofenriching.
 3. The cell-based diagnostic method of claim 2, furtherutilizing at least one cell attribute selected from the group consistingof a size, a shape, a structure, a form and an elasticity, whenutilizing said cell morphology based separation.
 4. The cell-baseddiagnostic method of claim 1, further trapping said one or more cells ata channel junction in said step (b) of sequestering.
 5. The cell-baseddiagnostic method of claim 4, further lysing said one or more cellsafter said trapping.
 6. The cell-based diagnostic method of claim 1,further performing nucleic amplification of said one or more cellsbefore said step (c) of sequencing.
 7. The cell-based diagnostic methodof claim 1, further barcoding said one or more cells after said step (b)of sequestering, and performing said step (c) of sequencing on barcodedcells.
 8. The cell-based diagnostic method of claim 1, further using agenetic variant information of said one or more cells, in said step (d)of determining of said provenance.
 9. The cell-based diagnostic methodof claim 8, wherein said genetic variant information is a singlenucleotide variant (SNV) information of said one or more cells.
 10. Thecell-based diagnostic method of claim 1, wherein said provenance is anelement selected from the group consisting of a maternal origin, a fetalorigin, a patient origin and a cancer origin.
 11. The cell-baseddiagnostic method of claim 1, further utilizing a consensus operation inanalyzing the data resulting from said step (c) of sequencing.
 12. Thecell-based diagnostic method of claim 1, further tagging individualmolecules of said one or more cells by unique barcodes.
 13. Thecell-based diagnostic method of claim 1, wherein said geneticcharacteristic is a trisomy status.
 14. The cell-based diagnostic methodof claim 1, wherein said diagnosis is an element selected from the groupconsisting of an aneuploidy, an inherited disease, a cancer, anauto-immune disease and an indication of an organ transplant rejection.15. The cell-based diagnostic method of claim 1, further providing atleast one element selected from the group consisting of a therapyguidance and a prognostic measure.
 16. A diagnostic apparatuscomprising: (a) a sequence data of at least one cell, said at least onecell lysed and separated from an enriched sub-population of cells; (b) aprovenance of said at least one cell, said provenance determined basedon said sequence data; (c) a genetic trait of said at least one cellbased on said sequence data, said genetic trait determined if saidprovenance is of a predetermined type; wherein a diagnostic readout isprovided based on said genetic trait.
 17. The diagnostic apparatus ofclaim 16, further comprising a barcode applied to said at least onecell.
 18. The diagnostic apparatus of claim 16, wherein said apparatusis used in an item selected from the group consisting of non-invasiveprenatal testing (NIPT), oncological testing, detection of anauto-immune disease and detection of an organ transplant rejection. 19.The diagnostic apparatus of claim 16, wherein said diagnostic readoutcomprises at least one element selected from the group consisting of ananeuploidy, a single nucleotide variant (SNV) abnormality, and a somaticmutation.
 20. A method of non-invasive pre-natal testing (NIPT)comprising the steps of: (a) enriching fetal cells in a blood sampledrawn from a pregnant mother; (b) separating at least one cell from saidblood sample after said enrichment; (c) applying a barcode to thenucleic acid of said at least one cell; (d) utilizing said barcode inidentifying and sequencing said at least one cell; and (e) determining agenetic trait of said at least one cell if said at least one cell isdetermined to be of fetal origin.